Field of the Invention
The present invention relates to the fields of biotechnology, and particularly to extracellular microvesicles such as exosomes. The invention provides surprising new methods and compositions for isolating disease-related and phosphatidylserine (PS)-positive extracellular microvesicles such as tumor- and viral-derived exosomes, particularly tumor-derived exosomes, which are especially suitable for use with large volumes of biological fluids and produce extracellular microvesicles and exosomes that are antigenically intact.
Description of the Related Art
Extracellular microvesicles are a class of membrane bound components released or secreted by cells, and include exosomes, ectosomes, microparticles or microvesicles and apoptotic bodies or blebs (György et al., 2011; Simpson & Mathivanan, 2012). Within this class of extracellular microvesicles, exosomes have gained particular attention in recent years.
Exosomes are typically described as 40-50 to 100 nanometer-sized membrane-derived vesicles and are known to be actively secreted by cells in vivo and in vitro. They are generated from the late endosomes by the inward budding and scission of the endosomal membrane, creating multivesicular bodies (MVBs) that contain intraluminal vesicles. These exosomes are released to the extracellular space upon fusion of the MVB with the plasma membrane. Because they originate from the cell's plasma membrane and are formed by invagination of the endosomal membrane, secreted exosomes possess plasma membrane and endosome proteins that encapsulate a cytosol-derived aqueous space.
Extracellular microvesicles such as exosomes exert a broad array of important physiological functions, e.g., by acting as molecular messengers that traffic information between different cell types. For example, exosomes deliver proteins, lipids and soluble factors including RNA and microRNAs (Thery et al., 2009) which, depending on their source, participate in signaling pathways that can influence apoptosis (Andreola et al., 2002; Huber et al., 2005; Kim et al., 2005), metastasis (Parolini et al., 2009), angiogenesis (Kim et al., 2005; Iero et al., 2008), tumor progression (Keller et al., 2009; Thery et al., 2002), thrombosis (Aharon & Brenner, 2009; Al Nedawi et al., 2005) and immunity by directing T cells towards immune activation (Andre et al., 2004; Chaput et al., 2005) or immune suppression (Szajnik et al., 2010; Valenti et al., 2007; Wieckowski et al., 2009).
Several techniques have been described for the isolation and purification of extracellular microvesicle and exosome populations from different sources, including from malignant effusions and the peripheral blood of cancer patients and from supernatants of in vitro cultivated cell lines and tumor cells. These methods include differential centrifugation, including an ultracentrifugation step (Thery et al., 2006); affinity chromatography (Taylor & Gercel-Taylor, 2008); polymer-mediated precipitation (Taylor et al., 2011), particularly using polyethylene glycol (PEG) of different molecular weights, including the Total Exosome Isolation Reagents from Life Technologies Corporation (U.S. Pat. No. 8,901,284) and ExoQuick™ (US 2013/0337440 A1); and capture on defined pore-size membranes (Grant et al., 2011), such as ExoMir™, which typically uses two filters of different pore-sizes connected in series (US 2013/0052647 A1).
However, the available techniques are limited by drawbacks in two important respects. Firstly, as applied to extracellular microvesicle and exosome preparation in general, they are time-consuming, cumbersome and/or costly, and limited by the amounts of material that can be processed. In particular, the techniques currently available for isolating extracellular microvesicles and exosomes all require a significant reduction in volume to obtain sufficient concentrations for study or use. The typical approach of concentrating the biological medium using ultracentrifugation before proceeding with exosome isolation is very time consuming and requires specialized laboratory equipment.
Secondly, the current techniques are particularly limited as they apply to tumor-derived extracellular microvesicle and exosomes. For example, the extra-corporeal removal of exosomes from the circulation of cancer patients has been proposed, in which patient's blood is pumped through a lectin-affinity column and then returned to the patient (U.S. Pat. No. 8,288,172). It has also been reported that tumor-derived exosomes can be purified using paramagnetic beads coated with antibodies against tumor-specific proteins such as HER2/neu (Koga et al., 2005). Kits using magnetic beads to capture specific exosomes are also available, such as Exo-Flow™ kits. In addition to the general drawbacks described above, such methods and kits are very limiting, requiring both advance knowledge of a particular exosome surface marker to be exploited in the antibody binding, as well as many detailed technical steps in the protocol, such as the preparation and use of biotinylated capture antibodies.
Therefore, there remains in the art a need for new and improved methods of isolating extracellular microvesicles such as exosomes, particularly disease-related and tumor-derived exosomes. The identification of simple and cost-effective new methods of isolating morphologically and antigenically intact extracellular microvesicles and exosomes would be an important advance. What is really needed is a method that is equipped to handle large volumes of biological materials, without specialized laboratory equipment and without the need for an early ultracentrifugation step, and particularly one that can be used to preferentially isolate disease-related and tumor-derived exosomes.